Soil microbial lipid biomarkers are indicators of viable microbial biomass and community structure. Pressurized liquid extraction (PLE) of soil phospholipid fatty acids (PLFA) and neutral lipid fatty acids (NLFA) was compared to a conventional extraction method in four soils with differing physical and chemical properties. PLE efficiency was greater than that of the conventional method for about half of the saturated PLFA and for selected other Gram-positive (i16:0) and Gram-negative bacteria (18:1omega7c) PLFA, fungal PLFA (18:2omega6,9c), and eukaryotic NLFA from a coarse-textured soil. Lipids extracted by the two methods did not indicate a significant difference in microbial community structure data. Principle component analysis revealed that PLFA clustered by location, with data indicating that the group of microbes contributing the greatest weight differed among soils. Overall, the PLE method proved to be more efficient at extracting soilborne microbial lipids while not altering microbial community information. These advantages indicate the PLE method is robust and well-suited to soil microbial ecology research.